Streptococcus pneumoniae is a gram positive organism that is responsible for human diseases such as pneumonia, sinusitis, meningitis and endocarditis. New vaccine and antibiotic targets are proteins that are responsible for metal accumulation. In addition, S. pneumoniae experiences a wide variety of metal concentrations in its host organism. Zinc is an essential metal for S. pneumonaie growth and potentially for virulence. It is the long-term objective of this study to understand zinc homeostasis in S. pneumoniae. The first aim of the study is to characterize the regulators that control the expression of zinc efflux and influx. The important residues for metal binding will be determined in AdcR (the regulator of zinc influx) using in vivo growth assays. The affinity of SczA (the regulator of zinc efflux) for zinc will be monitored using fluorescence competition assays. The second aim of the study will be the evaluation of zinc homeostasis. Mutations to critical components of zinc homeostasis in S. pneumoniae will be made. Evaluation of zinc uptake, intracellular concentration and gene expression will be made using ratiometric mass spectrometry, LC-MS, and gene array data. These studies will give us insights into how S. pneumoniae maintains homeostasis of zinc, and will lead to experiments to alter this homeostatic window and how these changes affect the homeostasis of other metals. The last aim of this study will be to elucidate the nature of metal selectivity between the solute binding proteins, AdcA and PsaA, which allow transport of zinc and manganese, respectively, into the cell. Chimeric proteins will be constructed that should alter the metal preference for each protein. Biophysical characterization, in vivo growth assays and ratiometric mass spectrometry will be performed to assess the ability of these modified proteins to discriminate between zinc and manganese. PUBLIC HEALTH RELEVANCE: Streptococcus pneumoniae is a gram positive organism that is responsible for human diseases such as pneumonia, sinusitis, meningitis and endocarditis. New vaccine and antibiotic targets are proteins that are responsible for metal accumulation. The goal of this study is to elucidate how Streptococcus pneumoniae handles zinc toxicity and deprivation to aide in the discovery of new vaccine targets for researchers.